Acetohydroxy acid synthase variant and a microorganism comprising the same

ABSTRACT

The present disclosure relates to a novel acetohydroxy acid synthase, a microorganism comprising the same, or a method for producing an L-branched-chain amino acid using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. applicationSer. No. 17/076,057 filed Oct. 21, 2020, now U.S. Pat. No. 11,085,029,which is a divisional of U.S. application Ser. No. 16/479,813 filed Jul.22, 2019, now U.S. Pat. No. 10,844,359, which is a U.S. national phaseapplication of PCT/KR2018/007821, filed Jul. 10, 2018, which claimspriority to KR Application No. 10-2017-0087978, filed Jul. 11, 2017.U.S. application Ser. Nos. 16/479,813 and 17/076,057 are hereinincorporated by reference in their entity.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 200187_4149D3_SEQUENCE_LISTING.txt. The textfile is 59 KB, was created on Jun. 23, 2021, and is being submittedelectronically via EFS-Web.

TECHNICAL FIELD

The present disclosure relates to a novel acetohydroxy acid synthasevariant and a use thereof, and specifically, to an acetohydroxy acidsynthase variant, a microorganism containing the variant, or a methodfor producing an L-branched-chain amino acid.

BACKGROUND ART

Branched-chain amino acids (e.g., L-valine, L-leucine, and L-isoleucine)are known to increase protein levels in an individual and have animportant role as an energy source during exercise, and thus are widelyused in medicines, foods, etc. With regard to the biosynthesis ofbranched-chain amino acids, the same enzymes are used in parallelbiosynthesis pathways, and thus it is difficult to produce a single kindof branched-chain amino acid on an industrial scale via fermentation. Inthe preparation of branched-chain amino acids, the role of acetohydroxyacid synthase (i.e., the first enzyme in the biosynthesis ofbranched-chain amino acids) is most important; however, previous studieson acetohydroxy acid synthase were mainly focused on release of feedbackinhibition due to modifications of acetohydroxy acid synthase smallsubunit (IlvN protein) (Protein Expr Purif. 2015 May; 109:106-12,US2014-0335574, US2009-496475, US2006-303888, US2008-245610), thusrevealing a serious lack of relevant studies.

Acetohydroxy acid synthase is an enzyme which has roles of producingacetolactic acid from two molecules of pyruvate and producing2-aceto-2-hydroxy-butyrate from ketobutyric acid and pyruvate. Theacetohydroxy acid synthase catalyzes decarboxylation of pyruvate and acondensation reaction with another pyruvate molecule to produceacetolactate, which is a precursor of valine and leucine; or catalyzesdecarboxylation of pyruvate and a condensation reaction with2-ketobutyrate to produce acetohydroxybutyrate, which is a precursor ofisoleucine. Accordingly, acetohydroxy acid synthase is a very importantenzyme involved in the initial biosynthesis process of L-branched-chainamino acids.

DISCLOSURE Technical Problem

The present inventors have made efforts for effective production ofL-branched-chain amino acids, and as a result, they have developed alarge subunit variant. Then, the present inventors confirmed thatL-branched-chain amino acids can be produced in high yield from amicroorganism containing the variant, thereby completing the presentdisclosure.

Technical Solution

An object of the present disclosure is to provide an acetohydroxy acidsynthase variant.

Another object of the present disclosure is to provide a polynucleotideencoding the acetohydroxy acid synthase variant, a vector containing thepolynucleotide, and a transformant in which the vector is introduced.

Still another object of the present disclosure is to provide amicroorganism producing an L-branched-chain amino acid, in which themicroorganism contains the acetohydroxy acid synthase variant or intowhich the vector is introduced.

Still another object of the present disclosure is to provide a methodfor producing an L-branched-chain amino acid, which includes: culturingthe microorganism producing the L-branched-chain amino acid in a medium;and recovering the L-branched-chain amino acid from the microorganism orcultured medium thereof.

Advantageous Effects

When the activity of the acetohydroxy acid synthase variant according tothe present disclosure is introduced into a microorganism, themicroorganism can significantly increase the ability to produce anL-branched-chain amino acid. Therefore, the microorganism can be widelyused for large-scale production of L-branched-chain amino acids.

BEST MODE

To achieve the above objects, an aspect of the present disclosureprovides an acetohydroxy acid synthase variant, in which, in theacetohydroxy acid synthase large subunit (i.e., acetolactate synthaselarge subunit; IlvB protein), the 96^(th) amino acid (i.e., threonine)is substituted with an amino acid other than threonine, the 503rd aminoacid (i.e., tryptophan) is substituted with an amino acid other thantryptophan, or both the 96^(th) amino acid (i.e., threonine) and the503^(rd) amino acid (i.e., tryptophan) are substituted with anotheramino acid.

Specifically, the large subunit of the acetohydroxy acid synthase mayhave an amino acid sequence of SEQ ID NO: 1. More specifically, theacetohydroxy acid synthase variant may be an acetohydroxy acid synthasevariant, in which, in the amino acid sequence of SEQ ID NO: 1, the96^(th) amino acid (i.e., threonine) or the 503rd amino acid (i.e.,tryptophan) from the N-terminus thereof is substituted with anotheramino acid; or both the 96^(th) amino acid (i.e., threonine) and the503^(rd) amino acid (i.e., tryptophan) are each substituted with anotheramino acid.

As used herein, the term “acetohydroxy acid synthase” refers to anenzyme involved in the biosynthesis of L-branched-chain amino acids, andit may be involved in the first step of the biosynthesis ofL-branched-chain amino acids. Specifically, acetohydroxy acid synthasemay catalyze decarboxylation of pyruvate and a condensation reactionwith another pyruvate molecule to produce acetolactate (i.e., aprecursor of valine) or may catalyze decarboxylation of pyruvate and acondensation reaction with 2-ketobutyrate to produceacetohydroxybutyrate (i.e., a precursor of isoleucine). Specifically,starting from acetolactic acid, L-valine is biosynthesized by sequentialreactions catalyzed by acetohydroxy acid isomeroreductase, dihydroxyacid dehydratase, and transaminase B. Additionally, starting fromacetolactic acid, L-leucine is biosynthesized as a final product bysequential reactions catalyzed by acetohydroxy acid isomeroreductase,dihydroxy acid dehydratase, 2-isopropylmalate synthase, isopropylmalateisomerase, 3-isopropylmalate dehydrogenase, and transaminase B.Meanwhile, starting from acetohydroxybutyrate, L-isoleucine isbiosynthesized as a final product by sequential reactions catalyzed byacetohydroxy acid isomeroreductase, dihydroxy acid dehydratase, andtransaminase B. Accordingly, acetohydroxy acid synthase is an importantenzyme in the biosynthesis pathway of L-branched-chain amino acids.

Acetohydroxy acid synthase is encoded by two genes, i.e., ilvB and ilvN.The ilvB gene encodes the large subunit of acetohydroxy acid synthase(IlvB), and the ilvN gene encodes the small subunit of acetohydroxy acidsynthase (IlvN).

In the present disclosure, the acetohydroxy acid synthase may be onederived from a microorganism of the genus Corynebacterium, andspecifically from Corynebacterium glutamicum. More specifically, as thelarge subunit of the acetohydroxy acid synthase, any protein having theIlvB protein activity and a homology or identity of 70% or higher,specifically 80% or higher, more specifically 85% or higher, even morespecifically 90% or higher, and even yet more specifically 95%, to theamino acid sequence of SEQ ID NO: 1 as well as the amino acid sequenceof SEQ ID NO: 1, may be included without limitation. Additionally, dueto codon degeneracy, the polynucleotide encoding the protein having theIlvB protein activity may be modified variously in the coding regionwithin a range that does not alter the amino acid sequence of theprotein expressed from the coding region, considering the codonspreferred in the organism for which the protein is to be expressed. Thenucleotide sequence may be included without limitation as long as itencodes the amino acid sequence of SEQ ID NO: 1, and specifically, itmay be one encoded by the nucleotide sequence of SEQ ID NO: 2.

As used herein, the term “acetohydroxy acid synthase variant” refers toa protein in which one or more amino acids are modified (e.g., added,deleted, or substituted) in the amino acid sequence of the acetohydroxyacid synthase protein. Specifically, the acetohydroxy acid synthasevariant is a protein in which its activity is effectively increasedcompared to its wild-type or before modification due to the modificationof the present disclosure.

As used herein, the term “modification” refers to a common method forimproving enzymes, and any method known in the art may be used withoutlimitation, including strategies such as rational design and directedevolution. For example, the strategies for rational design include amethod for specifying an amino acid at a particular position(site-directed mutagenesis or site-specific mutagenesis), etc., and thestrategies for directed evolution include a method for inducing randommutagenesis, etc. Additionally, the modification may be one induced bynatural mutation without external manipulation. Specifically, theacetohydroxy acid synthase variant may be one which is isolated, arecombinant protein, or one which has occurred non-naturally, but theacetohydroxy acid synthase variants are not limited thereto.

The acetohydroxy acid synthase variant of the present disclosure may bespecifically an IlvB protein having an amino acid sequence of SEQ ID NO:1, in which the 96^(th) amino acid (threonine) or the 503^(rd) aminoacid (tryptophan) from the N-terminus thereof is mutated; or both the96^(th) amino acid (threonine) and the 503^(rd) amino acid (tryptophan)are simultaneously substituted with another amino acid, but theacetohydroxy acid synthase variant is not limited thereto. For example,the acetohydroxy acid synthase variant of the present disclosure may bean IlvB protein, in which the 96^(th) amino acid (threonine) issubstituted with serine, cysteine, or alanine, or the 503^(rd) aminoacid (tryptophan) is substituted with glutamine, asparagine, or leucine.Additionally, it is apparent that any acetohydroxy acid synthase variantwhich has an amino acid sequence in which the 96^(th) amino acid or the503^(rd) amino acid is substituted with another amino acid, andsimultaneously, part of the amino acid sequence is deleted, modified,substituted, or added, may exhibit an activity which is identical orcorresponding to that of the acetohydroxy acid synthase variant of thepresent disclosure.

Furthermore, the large subunits themselves of the acetohydroxy acidsynthase variants with the modifications described above, acetohydroxyacid synthase including the large subunits of the acetohydroxy acidsynthase variants, and acetohydroxy acid synthase including both thelarge and small subunits of the acetohydroxy acid synthase variants mayall be included in the scope of the acetohydroxy acid synthase variantof the present disclosure, but the acetohydroxy acid synthase variant isnot limited thereto.

In the present disclosure, it was confirmed that the amount ofL-branched-chain amino acid production can be increased by thesubstitution of the 96^(th) amino acid and the 503^(rd) amino acid ofthe acetohydroxy acid synthase protein with various other amino acids,and thus it was confirmed that amino acid positions at 96 and 503 areimportant in the modification of the acetohydroxy acid synthase proteinin connection with the L-branched-chain amino acid production. However,since the substituted amino acids in embodiments of the presentdisclosure are merely representative embodiments showing the effects ofthe present disclosure, the scope of the present disclosure should notbe limited to these embodiments, and it is apparent that when the96^(th) amino acid (threonine) is substituted with an amino acid otherthan threonine, the 503^(rd) amino acid (tryptophan) is substituted withan amino acid other than tryptophan, or both the 96^(th) amino acid andthe 503^(rd) amino acid are substituted with a different amino acid, theacetohydroxy acid synthase variants may have effects corresponding tothose described in embodiments.

Additionally, the acetohydroxy acid synthase variant of the presentdisclosure may have an amino acid sequence represented by any one of SEQID NOS: 28 to 33, but the amino acid sequence of the acetohydroxy acidsynthase variant is not limited thereto. Additionally, any polypeptidewhich has a homology or identity of at least 70%, at least 80%, at least85%, at least 90%, at least 95%, or at least 99% to the above amino acidsequences may also be included without limitation, as long as thesepolypeptides have an activity substantially identical or correspondingto that of the acetohydroxy acid synthase variant by including themodifications of the present disclosure.

Homology and identity refer to a degree of relatedness between two givenamino acid sequences or nucleotide sequences and may be expressed as apercentage.

The terms “homology” and “identity” may often be used interchangeablywith each other.

Sequence homology or identity of a conserved polynucleotide orpolypeptide may be determined by a standard alignment algorithm, anddefault gap penalties established by a program to be used may be used incombination. Substantially, homologous or identical sequences mayhybridize under moderately or highly stringent conditions along theirentire sequence or at least about 50%, about 60%, about 70%, about 80%,or about 90% of the entire length. With regard to the polynucleotides tobe hybridized, polynucleotides including a degenerate codon instead of acodon may also be considered.

Whether any two polynucleotide or polypeptide sequences have homology,similarity, or identity may be determined by, for example, a knowncomputer algorithm such as the “FASTA” program using default parametersas in Pearson et al. (1988) (Proc. Natl. Acad. Sci. USA 85]: 2444).Alternatively, they may be determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443 to 453) asperformed in the Needleman program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends Genet. 16: 276 to 277) (version 5.0.0 or later) (including GCGprogram package (Devereux, J., et al., Nucleic Acids Research 12: 387(1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.] et al., J Molec Biol215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,]Academic Press, San Diego, 1994, and [CARILLO ETA/.](1988) SIAM JApplied Math 48: 1073). For example, homology, similarity, or identitymay be determined using BLAST or ClustalW of the National Center forBiotechnology Information.

Homology, similarity, or identity of polynucleotides or polypeptides maybe determined by comparing sequence information using the GAP computerprogram (e.g., Needleman et al. (1970), J Mol Biol 48: 443) as disclosedin Smith and Waterman, Adv. Appl. Math (1981) 2:482. Briefly, the GAPprogram defines similarity as the number of aligned symbols (i.e.,nucleotides or amino acids) which are similar, divided by the totalnumber of symbols in the shorter of the two sequences. The defaultparameters for the GAP program may include: (1) a unary comparisonmatrix (containing a value of 1 for identities and 0 for non-identities)and the weighted comparison matrix (or EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix) of Gribskov et al. (1986) Nucl. Acids Res.14: 6745, as disclosed by Schwartz and Dayhoff, eds., Atlas Of ProteinSequence And Structure, National Biomedical Research Foundation, pp. 353to 358 (1979); (2) a penalty of 3.0 for each gap and an additional 0.10penalty for each symbol in each gap (or gap open penalty 10, gapextension penalty 0.5); and (3) no penalty for end gaps. Therefore, theterm “homology” or “identity”, as used herein, represents relevancebetween sequences.

Another aspect of the present disclosure provides a polynucleotideencoding the acetohydroxy acid synthase variant of the presentdisclosure.

As used herein, the term “polynucleotide” has a meaning to include a DNAor RNA molecule, and a nucleotide, which is a basic building blockthereof, includes not only a natural nucleotide but also an analog inwhich a saccharide or base region is modified. In the presentdisclosure, the polynucleotide may be a polynucleotide isolated from acell or an artificially synthesized polynucleotide, but thepolynucleotide is not limited thereto.

The polynucleotide encoding the acetohydroxy acid synthase variant ofthe present disclosure may include without limitation any nucleotidesequence that encodes the protein having an activity of the acetohydroxyacid synthase variant of the present disclosure. Specifically, due tocodon degeneracy or in consideration of the codons preferred by amicroorganism in which the protein is to be expressed, variousmodifications may be made in the coding region of the protein within thescope that does not change the amino acid sequence of the protein. Thepolynucleotide may include without limitation any nucleotide sequenceencoding the amino acid sequence of SEQ ID NOS: 28 to 33, andspecifically, one having the nucleotide sequence of SEQ ID NOS: 34 to39. Additionally, any polypeptide which has a homology or identity of atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 97%, or at least 99% to the above amino acidsequences may also be included without limitation, as long as thesepolypeptides have an activity substantially identical or correspondingto that of the acetohydroxy acid synthase variant by including themodifications of the present disclosure due to codon degeneracy.

Alternatively, by hybridization under stringent conditions with a probethat can be prepared from a known gene sequence (e.g., a sequencecomplementary to all or part of the nucleotide sequence), any sequenceencoding a protein having the activity of the proteins consisting of theamino acid sequence of SEQ ID NOS: 28 to 33 may be included withoutlimitation.

The “stringent conditions” refer to conditions that enable specifichybridization between polynucleotides. Such conditions are described indetail in the literature (e.g., J. Sambrook et al., supra). Thestringent conditions may include conditions under which genes havinghigh homology or identity (e.g., genes having at least 80%, specificallyat least 85%, more specifically at least 90%, even more specifically atleast 95%, even yet more specifically at least 97%, or most specificallyat least 99%) can hybridize to each other; conditions under which geneshaving lower homology or identity cannot hybridize to each other; orconditions which are common washing conditions for Southernhybridization (e.g., a salt concentration and a temperaturecorresponding to 60° C., 1×SSC, 0.1% SDS; specifically 60° C., 0.1×SSC,0.1% SDS; more specifically 68° C., 0.1×SSC, 0.1% SDS, once,specifically, twice or three times).

Hybridization requires that two nucleic acids have complementarysequences, although mismatches between bases may be possible dependingon hybridization stringency. The term “complementary” is used todescribe the relationship between nucleotide bases that can hybridize toeach another. For example, with respect to DNA, adenosine iscomplementary to thymine, and cytosine is complementary to guanine.Accordingly, the present disclosure may also include isolated nucleicacid fragments complementary to the entire sequence as well as tosubstantially similar nucleic acid sequences.

Specifically, a polynucleotide having homology or identity may bedetected using hybridization conditions including a hybridization stepat T_(m) of 55° C. and by utilizing the above-described conditions.Additionally, the T_(m) value may be 60° C., 63° C., or 65° C., but isnot limited thereto, and may be appropriately controlled by thoseskilled in the art according to the purpose. The appropriate stringencyfor hybridizing polynucleotides depends on the length of thepolynucleotides and the degree of complementarity, and variables arewell known in the art (see Sambrook et al., supra, 9.50 to 9.51, 11.7 to11.8).

Still another aspect of the present disclosure provides a vectorincluding the polynucleotide encoding the modified acetohydroxy acidsynthase variant of the present disclosure.

As used herein, the term “vector” refers to any carrier for cloningand/or transferring nucleotides into a host cell. A vector may be areplicon to allow for the replication of a fragment(s) combined withother DNA fragment(s). “Replicon” refers to any genetic unit functioningas a self-replicating unit for DNA replication in vivo, that is,replicable by self-regulation. Specifically, the vector may be plasmids,phages, cosmids, chromosomes, or viruses in a natural or recombinedstate. For example, as a phage vector or cosmid vector, pWE15, M13,λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, Charon21A,etc. may be used, and as a plasmid vector, those based on pBR, pUC,pBluescriptll, pGEM, pTZ, pCL, pET, etc. may be used. The vectors thatcan be used in the present disclosure are not particularly limited, butany known expression vector may be used. Additionally, the vector mayinclude a transposon or artificial chromosome.

In the present disclosure, the vector is not particularly limited aslong as it includes a polynucleotide encoding the acetohydroxy acidsynthase variant of the present disclosure. The vector may be one whichcan replicate and/or express the nucleic acid molecule in eukaryotic orprokaryotic cells including mammalian cells (e.g., cells of humans,monkeys, rabbits, rats, hamsters, mice, etc.), plant cells, yeast cells,insect cells, and bacteria cells (e.g., E. coli, etc.), andspecifically, may be one which is operably linked to a suitable promoterso that the polynucleotide can be expressed in a host cell and includeat least one selectable marker.

Additionally, as used herein, the term “operably linked” refers to afunctional connection between a promoter sequence, which initiates andmediates the transcription of the polynucleotide encoding the targetprotein of the present disclosure, and the above gene sequence.

Still another aspect of the present disclosure provides a transformantinto which the vector of the present disclosure is introduced.

In the present disclosure, the transformant may be any transformablecell into which the above vector can be introduced and in which theacetohydroxy acid synthase variant of the present disclosure can beexpressed. Specifically, the transformant may be any transformed cellsof bacteria belonging to the genus Escherichia, the genusCorynebacterium, the genus Streptomyces, the genus Brevibacterium, thegenus Serratia, the genus Providencia, Salmonella typhimurium, etc.;cells of yeasts; fungal cells of Pichia pastoris, etc.; transformedcells of insects (e.g., Drosophila, Spodoptera Sf9, etc.); andtransformed animal cells (e.g., cells of Chinese hamster ovary (CHO),SP2/0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma),293T, bow melanoma, HT-1080, baby hamster kidney (BHK), human embryonickidney (HEK), PERC.6 (human retinocytes)); or transformed plant cells,but the transformant is not particularly limited thereto.

Still another aspect of the present disclosure provides a microorganismproducing L-branched chain amino acids, in which the microorganismcontains the acetohydroxy acid synthase variant or into which a vectorcontaining a polynucleotide encoding the variant is introduced.

As used herein, the term “L-branched-chain amino acid” refers to anamino acid with a branched alkyl group on the side chain, and itincludes valine, leucine, and isoleucine. Specifically, in the presentdisclosure, the L-branched-chain amino acid may be L-valine orL-leucine, but is not limited thereto.

As used herein, the term “microorganism” includes all of a wild-typemicroorganism and a naturally or artificially genetically modifiedmicroorganism, and it is a concept including all of the microorganismsin which a particular mechanism is attenuated or enhanced due toinsertion of an exogenous gene or enhancement or attenuation of activityof an endogenous gene. The microorganism refers to all of themicroorganisms that can express the acetohydroxy acid synthase variantof the present disclosure. Specifically, the microorganism may beCorynebacterium glutamicum, Corynebacterium ammoniagenes, Brevibacteriumlactofermentum, Brevibacterium flavum, Corynebacterium thermoaminogenes,Corynebacterium efficiens, etc., and more specifically Corynebacteriumglutamicum, but the microorganism is not limited thereto.

As used herein, the term “microorganism producing L-branched-chain aminoacids” may refer to a natural microorganism or a modified microorganismwhich has the ability to produce L-branched-chain amino acids viamodification, and specifically may refer to a non-naturally occurringrecombinant microorganism, but the microorganism is not limited thereto.The microorganism producing L-branched-chain amino acids is amicroorganism which contains the acetohydroxy acid synthase variant ofthe present disclosure or into which a vector containing apolynucleotide encoding the variant is introduced, and the microorganismmay have a significantly increased ability to produce L-branched-chainamino acids compared to a wild-type microorganism, a microorganismcontaining a natural-type acetohydroxy acid synthase protein, anon-modified microorganism containing a acetohydroxy acid synthaseprotein, and a microorganism not containing a acetohydroxy acid synthaseprotein.

Still another aspect of the present disclosure provides a method forproducing L-branched-chain amino acids, which includes: culturing amicroorganism producing L-branched-chain amino acids; and recovering theL-branched-chain amino acids from the microorganism or cultured mediumin the above step.

As used herein, the term “culture” refers to culturing of amicroorganism under artificially controlled environmental conditions. Inthe present disclosure, the method for producing an L-branched-chainamino acid using a microorganism capable of producing anL-branched-chain amino acid may be carried out by a method widely knownin the art. Specifically, the culture may be carried out in a batchprocess, fed-batch process, or repeated fed-batch process, but the batchprocess is not limited thereto.

The medium used for the culture must satisfy the requirements of aparticular strain used. For example, the culture medium suitable for usein culturing the Corynebacterium strain is known in the art (e.g.,Manual of Methods for General Bacteriology by the American Society forBacteriology, Washington D.C., USA, 1981).

Saccharide sources that can be used in the culture medium may besaccharides and carbohydrates (e.g., glucose, sucrose, lactose,fructose, maltose, starch, and cellulose); oils and lipids (e.g.,soybean oil, sunflower seed oil, peanut oil, and coconut oil); fattyacids (e.g., palmitic acid, steric acid, and linoleic acid); alcohols(e.g., glycerol and ethanol); and organic acids (e.g., acetic acid).These materials may be used independently or in combination, but themodes of use are not limited thereto.

Examples of nitrogen sources that can be used in the culture medium mayinclude peptone, yeast extract, meat juice, malt extract, corn steepliquor, soybean meal, and urea, or inorganic compounds (e.g., ammoniumsulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, andammonium nitrate). These nitrogen sources may also be used independentlyor in combination, but the modes of use are not limited thereto.

Phosphorous sources that can be used in the culture medium may includepotassium dihydrogen phosphate, dipotassium hydrogen phosphate, orcorresponding sodium-containing salts. In addition, the culture mediummay contain metal salts necessary for the growth of cells. Further, inaddition to the materials above, materials essential for growth (e.g.,amino acids and vitamins) may be used. Additionally, precursors suitablefor the culture medium may be used. The above raw materials may beadequately added into the culture during the culture process in a batchor continuous manner, but the method of addition is not limited thereto.

The pH of the culture may be adjusted using a basic compound (e.g.,sodium hydroxide, potassium hydroxide, or ammonia) or an acidic compound(e.g., phosphoric acid or sulfuric acid) in an appropriate manner.Additionally, foam generation may be prevented using an anti-foamingagent (e.g., fatty acid polyglycol ester). Oxygen or oxygen-containinggas (e.g., air) may be injected into the culture so as to maintain theaerobic condition of the culture. The temperature of the culture may begenerally in a range of 20° C. to 45° C., and specifically 25° C. to 40°C. Culturing may be continued until the maximum amount of theL-branched-chain amino acid is produced, and specifically for 10 to 160hours. The L-branched-chain amino acid may be released into the culturemedium or contained in the cells, but is not limited thereto.

The method of recovering an L-branched-chain amino acid from amicroorganism or culture may include those well known in the art; forexample, centrifugation, filtration, treatment with a proteincrystallizing precipitant (salting-out method), extraction, ultrasonicdisruption, ultrafiltration, dialysis, various kinds of chromatographies(e.g., molecular sieve chromatography (gel filtration), adsorptionchromatography, ion-exchange chromatography, affinity chromatography,etc.), HPLC, and a combination thereof may be used, but the methods arenot limited thereto. Additionally, the step of recovering theL-branched-chain amino acid may further include a purification process,and the purification process can be performed using an appropriatemethod known in the art.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present disclosure will be described in more detailwith reference to the following Examples. However, these Examples arefor illustrative purposes only, and the present disclosure is notintended to be limited by these Examples.

Example 1: Preparation of Library of DNA Encoding Modified AcetohydroxyAcid Synthase Using Artificial Mutagenesis

In this Example, a vector library for primary crossover-insertion withinthe chromosome for obtaining acetohydroxy acid synthase variants wasprepared by the following method. Error-prone PCR was performed for ilvBgene (SEQ ID NO: 2) encoding acetohydroxy acid synthase (SEQ ID NO: 1)derived from Corynebacterium glutamicum ATCC14067, and thereby ilvB genevariants (2,395 bp) of ilvB gene variants randomly introduced with amutation(modification) of nucleotide substitution were obtained. Theerror-prone PCR was performed using the GenemorphII Random MutagenesisKit (Stratagene), using the genomic DNA of Corynebacterium glutamicumATCC14067 as a template along with primer 1 (SEQ ID NO: 3) and primer 2(SEQ ID NO: 4).

primer 1 (SEQ ID NO: 3): 5′-AACCG GTATC GACAA TCCAA T-3′primer 2 (SEQ ID NO: 4): 5′-GGGTC TCTCC TTATG CCTC-3′

The error-prone PCR was performed such that modifications can beintroduced into the amplified gene fragment at a ratio of 0 to 3.5mutations per 1 kb of the amplified gene fragment. PCR was performed fora total of 30 cycles as follows: denaturation at 96° C. for 30 sec,annealing at 53° C. for 30 sec, and polymerization at 72° C. for 2 min.

The amplified gene fragments were connected to the pCR2.1-TOPO vector(hereinafter, “pCR2.1”) using the pCR2.1-TOPO TA Cloning Kit(Invitrogen), transformed into E. coli DH5a, and plated on a solid LBmedium containing kanamycin (25 mg/L). 20 of the transformed colonieswere selected, and their nucleotide sequences were analyzed afterobtaining their plasmids. As a result, it was confirmed thatmodifications were introduced at different locations at a frequency of2.1 mutations/kb. Plasmids were extracted from about 20,000 transformedE. coli colonies, and they were named “pCR2.1-ilvB(mt) library”.

Additionally, a plasmid including the wild-type ilvB gene to be used asa control was prepared. PCR was performed using the genomic DNA ofCorynebacterium glutamicum ATCC14067 as a template along with primer 1(SEQ ID NO: 3) and primer 2 (SEQ ID NO: 4), under the same conditionsdescribed above. For the polymerase, PfuUltra™ high-fidelity DNApolymerase (Stratagene) was used, and the prepared plasmid was named“pCR2.1-ilvB(WT)”.

Example 2: Preparation of ilvB-Deficient Strain

An ilvB-deficient strain for the introduction of the pCR2.1-ilvB(mt)library was prepared using the KCCM11201P strain (KR Patent No.10-1117022) as the parent strain.

To prepare an ilvB-deficient vector, PCR was performed using thechromosomal DNA of the wild-type Corynebacterium glutamicum ATCC14067 asa template and a primer set of primer 3 (SEQ ID NO: 5) and primer 4 (SEQID NO: 6) and a primer set of primer 5 (SEQ ID NO: 7) and primer 6 (SEQID NO: 8).

primer 3 (SEQ ID NO: 5): 5′-GCGTC TAGAG ACTTG CACGA GGAAA CG-3′primer 4 (SEQ ID NO: 6):5′-CAGCC AAGTC CCTCA GAATT GATGT AGCAA TTATC C-3′primer 5 (SEQ ID NO: 7):5′-GGATA ATTGC TACAT CAATT CTGAG GGACT TGGCT G-3′primer 6 (SEQ ID NO: 8): 5′-GCGTC TAGAA CCACA GAGTC TGGAG CC-3′

PCR was performed as follows: denaturation at 95° C. for 5 min; 30cycles of denaturation at 95° C. for 30 sec, annealing at 55° C. for 30sec, and polymerization at 72° C. for 30 sec; and polymerization at 72°C. for 7 min.

As a result, a 731 bp DNA fragment (SEQ ID NO: 9), which includes theupstream region of the promoter of ilvB gene, and a 712 bp DNA fragment(SEQ ID NO: 10), which includes the 3′ terminus of the ilvB gene, wereobtained.

PCR was performed using the amplified DNA fragments (SEQ ID NOS: 9 and10) and a primer set of primer 3 (SEQ ID NO: 5) and primer 6 (SEQ ID NO:8). PCR was performed as follows: denaturation at 95° C. for 5 min; 30cycles of denaturation at 95° C. for 30 sec, annealing at 55° C. for 30sec, and polymerization at 72° C. for 60 sec; and polymerization at 72°C. for 7 min.

As a result, a 1,407 bp DNA fragment (SEQ ID NO: 11, hereinafter “ilvBfragment”), in which a DNA fragment including the upstream region of thepromoter of ilvB gene and a DNA fragment including the 3′ terminus ofthe ilvB gene are linked, was amplified.

The pDZ vector (KR Patent No. 10-0924065), which cannot replicate inCorynebacterium glutamicum, and the ilvB gene fragment amplified abovewere each treated with restriction enzyme XbaI, ligated using a DNAligase, and cloned. The obtained plasmid was named “pDZ-ilvB”.

The pDZ-ilvB was transformed into Corynebacterium glutamicum KCCM11201Pby the electroporation method (Appl. Microbiol. Biothcenol. (1999) 52:541-545), and the transformed strains were obtained in selection mediacontaining kanamycin (25 mg/L) and 2 mM each of L-valine, L-leucine, andL-isoleucine. The strain, in which the gene is inactivated by the ilvBgene fragment inserted into the genome during the secondary crossoverprocess, was obtained, and the strain was named KCCM11201PilvB.

Example 3: Preparation of Library of Modified Strains of AcetohydroxyAcid Synthase and Selection of Strains with Increased Ability ofProducing L-Amino Acids

The above-prepared KCCM11201PilvB strain was transformed by homologousrecombination using the above-prepared pCR2.1-ilvB(mt) library, and thetransformant was plated on a complex plate medium containing kanamycin(25 mg/L) and about 10,000 colonies were obtained therefrom. Thecolonies were named KCCM11201PilvB/pCR2.1-ilvB(mt)-1 toKCCM11201PilvB/pCR2.1-ilvB(mt)-10000.

Additionally, the above-prepared pCR2.1-ilvB(WT) vector was transformedinto the KCCM11201PilvB strain to prepare a control strain and thestrain was named KCCM11201PilvB/pCR2.1-ilvB(WT).

<Complex Plate Medium (pH 7.0)>

Glucose (10 g), Peptone (10 g), Beef Extract (5 g), Yeast Extract (5 g),Brain Heart Infusion (18.5 g), NaCl (2.5 g), Urea (2 g), Sorbitol (91g), Agar (20 g) (based on 1 L of distilled water)

About 25,000 colonies obtained above were each inoculated into aselective medium (300 μL) containing the components described below andcultured in a 96-deep well plate at 32° C. at a rate of 1,000 rpm for 24hours. The amounts of L-amino acids produced in the culture wereanalyzed by the ninhydrin method (J. Biol. Chem. 1948. 176: 367-388).Upon completion of the cultivation, 10 μL of the culture supernatant and190 μL of a ninhydrin reaction solution were reacted at 65° C. for 30minutes. The absorbance was measured at wavelength 570 nm using aspectrophotometer and was compared to that of the control, i.e.,KCCM11201PilvB/pCR2.1-ilvB(WT), and about 213 modified strains showingan absorbance with an at least 10% increase were selected. Othercolonies showed similar or reduced absorbance compared to that of thecontrol.

<Selective Medium (pH 8.0)>

Glucose (10 g), (NH₄)₂SO₄ (5.5 g), MgSO₄.7H₂O (1.2 g), KH₂PO₄ (0.8 g),K₂HPO₄ (16.4 g), Biotin (100 μg), Thiamine HCl (1,000 μg),Calcium-Pantothenic Acid (2,000 μg), and Nicotinamide (2,000 μg) (basedon 1 L of distilled water)

The above method was repeatedly performed for the selected 213 strains,and the top 60 kinds of strains with an improved ability of producingL-amino acids compared to that of KCCM11201PilvB/pCR2.1-ilvB(WT) wereselected.

Example 4: Confirmation of L-Valine-Producing Ability of StrainsSelected from the Library of Modified Strains of Acetohydroxy AcidSynthase

The 60 kinds of strains selected in Example 3 were analyzed with respectto their L-valine-producing abilities after culturing them by thefollowing method.

Each of the strains was inoculated into a 250 mL corner-baffle flaskcontaining 25 mL of a production medium, respectively, and cultured in ashaking incubator (200 rpm) at 30° C. for 20 hours. Then, each of the250 mL corner-baffle flasks containing 24 mL of the culture, whichcontained the components described below, was inoculated with 1 mL of aseed culture broth, and cultured with shaking (200 rpm) at 30° C. for 72hours. The concentration of L-valine in each culture was analyzed byHPLC.

<Production Medium (pH 7.0)>

Glucose (100 g), (NH₄)₂SO₄ (40 g), Soybean Protein (2.5 g), Corn SteepSolids (5 g), Urea (3 g), KH₂PO₄ (1 g), MgSO₄.7H₂O (0.5 g), Biotin (100μg), Thiamine HCl (1,000 μg), Calcium-Pantothenic Acid (2,000 μg),Nicotinamide (3,000 μg), and CaCO₃ (30 g) (based on 1 L of distilledwater)

Among the selected 60 kinds of strains, 2 kinds of strains showing anincrease in L-valine concentration were selected, and the cultivationand analysis were performed repeatedly. The analysis results of theL-valine concentration are shown in Table 1 below. The remaining 58kinds of strains actually showed a decrease in L-valine concentration.

TABLE 1 Concentration of L-Valine Produced by Two Selected Strains ofKCCM11201PilvB/pCR2.1-ilvB(mt) L-Valine (g/L) Strain Batch 1 Batch 2Batch 3 Mean Control KCCM11201PilvB/pCR2.1- 2.7 2.9 2.9 2.8 ilvB(WT) 1KCCM11201PilvB/pCR2.1- 3.1 3.5 3.4 3.3 ilvB(mt)-5602 2KCCM11201PilvB/pCR2.1- 2.9 3.3 3.1 3.1 ilvB(mt)-7131

As a result of the analysis of the L-valine concentration of the 2selected strains, it was confirmed that the L-valine yield of the twostrains was increased by 20.7% at maximum compared to that of thecontrol strain, KCCM11201PilvB/pCR2.1-ilvB(WT).

Example 5: Confirmation of ilvB Gene Modification in Strains Selectedfrom a Library of Modified Strains of Acetohydroxy Acid Synthase

To confirm the random modifications introduced into the acetohydroxyacid synthase of the 2 selected strains in Example 4, the nucleotidesequences of ilvB gene were analyzed. For determining the nucleotidesequences, PCR was performed using a primer set of primer 7 (SEQ ID NO:12) and primer 8 (SEQ ID NO: 13).

primer 7 (SEQ ID NO: 12): 5′-CGCTT GATAA TACGC ATG-3′primer 8 (SEQ ID NO: 13): 5′-GAACA TACCT GATAC GCG-3′

The obtained modified ilvB gene fragments were each subjected tonucleotide sequence analysis, and the results were compared to thenucleotide sequence of wild-type ilvB gene (i.e., SEQ ID NO: 2). As aresult, the nucleotide sequences of modified ilvB gene were confirmed,and the amino acid sequences of modified acetohydroxy acid synthaseproteins were confirmed. The information of the selected two kinds ofmodified acetohydroxy acid synthase proteins is shown in Table 2 below.

TABLE 2 Information of Selected Two Kinds of Modified Acetohydroxy AcidSynthase Proteins of KCCM11201P/pCR2.1-ilvB(mt) Amino Acid Modificationof Acetohydroxy Acid Strain Synthase KCCM11201PilvB/pCR2.1-ilvB(mt)-5602W503Q KCCM11201PilvB/pCR2.1-ilvB(mt)-7131 T96S

Example 6: Preparation of Vector for Introducing Modification inAcetohydroxy Acid Synthase

To confirm the effects of the modified acetohydroxy acid synthaseproteins which were confirmed in Example 5, a vector capable ofintroducing the modified acetohydroxy acid synthase proteins onto thechromosome was prepared.

Based on the confirmed nucleotide sequences, a primer set of the primer9 (SEQ ID NO: 14) and the primer 10 (SEQ ID NO: 15) and a primer set ofthe primer 11 (SEQ ID NO: 16) and the primer 12 (SEQ ID NO: 17), inwhich an XbaI restriction site was inserted at the 5′ end, weresynthesized. Then, PCR was performed using each of the selected twokinds of chromosomal DNAs as a template using these primer sets, andthereby the modified ilvB gene fragments were amplified. PCR wasperformed as follows: denaturation at 94° C. for 5 min; 30 cycles ofdenaturation at 94° C. for 30 sec, annealing at 56° C. for 30 sec, andpolymerization at 72° C. for 2 min; and polymerization at 72° C. for 7min.

primer 9 (SEQ ID NO: 14): 5′-CGCTC TAGAC AAGCA GGTTG AGGTT CC-3′primer 10 (SEQ ID NO: 15): 5′-CGCTC TAGAC ACGAG GTTGA ATGCG CG-3′primer 11 (SEQ ID NO: 16): 5′-CGCTC TAGAC CCTCG ACAAC ACTCA CC-3′primer 12 (SEQ ID NO: 17): 5′-CGCTC TAGAT GCCAT CAAGG TGGTG AC-3′

The two kinds of gene fragments amplified by PCR were treated with XbaIto obtain the respective DNA fragments, and linked these fragments tothe pDZ vector for chromosomal introduction, which includes an XbaIrestriction site therein, transformed into E. coli DH5α, and thetransformants were spread on an LB solid medium containing kanamycin (25mg/L).

The colonies transformed with a vector inserted with a target gene wereselected by PCR, and the plasmids were obtained by a commonly knownplasmid extraction method. These plasmids were named pDZ-ilvB(W503Q) andpDZ-ilvB(T96S), each according to the modifications inserted into theilvB gene.

Example 7: Preparation of KCCM11201P-Derived Strains with Modificationin Acetohydroxy Acid Synthase and Comparison of their L-Valine-ProducingAbilities

The two kinds of vectors introduced with novel modifications prepared inExample 6 were each transformed into the Corynebacterium glutamicumKCCM11201P, which is a strain producing L-valine, by a two-stephomologous chromosome recombination. Then, the strains introduced withthe ilvB gene modification on the chromosome were selected by theanalysis of nucleotide sequences. The strains introduced with the ilvBgene modification were named KCCM11201P::ilvB(W503Q) andKCCM11201P::ilvB(T96S). Additionally, the pDZ-ilvB(T96S) vector, betweenthe vectors introduced with the above modification, was transformed intothe KCCM11201P::ilvB(W503Q) strain prepared above. Then, the strainsinto which the two kinds of modifications on the chromosome wereintroduced were named KCCM11201P::ilvB(W503Q/T96S).

The strains were cultured in the same manner as in Example 4, and theL-valine concentrations were analyzed from the cultured strains (Table3).

TABLE 3 Concentration of L-Valine Produced by KCCM11201P-Derived StrainsIntroduced with Modified Acetohydroxy Acid Synthase (g/L) Strain Batch 1Batch 2 Batch 3 Mean Control KCCM11201P 2.9 2.8 2.8 2.8 1KCCM11201P::ilvB 3.3 3.2 3.3 3.3 (W503Q) 2 KCCM11201P::ilvB 3.2 3.0 3.13.1 (T96S) 3 KCCM11201P::ilvB 3.3 3.4 3.4 3.4 (W503Q/T96S)

As a result, two novel strains introduced with modifications(KCCM11201P::ilvB(W503Q) and KCCM11201P::ilvB(T96S)) showed a maximumincrease of 17.8% in L-valine-producing ability compared to the parentstrain, and the strain introduced with both modifications(KCCM11201P::ilvB(W503Q/T96S) showed an increase of 21.4% inL-valine-producing ability compared to the parent strain.

Accordingly, considering that acetohydroxy acid synthase is the firstenzyme in the biosynthesis pathways of L-branched-chain amino acids, theacetohydroxy acid synthase large subunit variants of the presentdisclosure are expected to have an effect on the production increase ofL-isoleucine and L-leucine as well as L-valine.

The present inventors have named the strains with an improved ability ofL-valine production (i.e., KCCM11201P::ilvB(W503Q) andKCCM11201P::ilvB(T96S)) as Corynebacterium glutamicum KCJ-0793 andCorynebacterium glutamicum KCJ-0796, and deposited them with the KoreanCulture Center of Microorganisms (KCCM) on Jan. 25, 2016, under theAccession Numbers KCCM11809P and KCCM11810P.

Example 8: Preparation of Overexpression Vector for L-ValineBiosynthesis Containing DNA Encoding Modified Acetohydroxy Acid Synthase

As a control group, an overexpression vector for L-valine biosynthesiswas prepared from Corynebacterium glutamicum KCCM11201P, which is astrain producing L-valine. Additionally, overexpression vectors forL-valine biosynthesis, in which DNAs encoding acetohydroxy acid synthasemodified from each of KCCM11201P::ilvB(W503Q) and KCCM11201P::ilvB(T96S)prepared in Example 7 are included, were prepared.

For the preparation of the above vectors, the primer 13 (SEQ ID NO: 18),in which a BamHI restriction site was inserted at the 5′ end, and theprimer 14 (SEQ ID NO: 19), in which an XbaI restriction site wasinserted at the 3′ end, were synthesized. Using the primer set, PCR wasperformed using each of the chromosomal DNAs of Corynebacteriumglutamicum KCCM11201P (i.e., a strain producing L-valine) and thestrains prepared in Example 7 (i.e., KCCM11201P::ilvB(W503Q) andKCCM11201P::ilvB(T96S)) as a template, and thereby two kinds of modifiedilvBN gene fragments were amplified. PCR was performed as follows:denaturation at 94° C. for 5 min; 30 cycles of denaturation at 94° C.for 30 sec, annealing at 56° C. for 30 sec, and polymerization at 72° C.for 4 min; and polymerization at 72° C. for 7 min.

primer 13 (SEQ ID NO: 18): 5′-CGAGG ATCCA ACCGG TATCG ACAAT CCAAT-3′primer 14 (SEQ ID NO: 19): 5′-CTGTC TAGAA ATCGT GGGAG TTAAA CTCGC-3′

The two kinds of gene fragments amplified by PCR were treated with BamHIand XbaI to obtain their respective DNA fragments. These DNA fragmentswere linked to the pECCG117 overexpression vector having BamHI and XbaIrestriction sites, transformed into E. coli DH5α, and plated on a solidLB medium containing kanamycin (25 mg/L).

The colonies transformed with a vector inserted with a target gene wereselected by PCR and the plasmids were obtained by a commonly knownplasmid extraction method. These plasmid were named pECCG117-ilvBN,pECCG117-ilvB(W503Q)N, and pECCG117-ilvB(T96S)N, each according to themodifications inserted into the ilvB gene.

Example 9: Preparation of Overexpression Vector for L-ValineBiosynthesis Containing DNA Encoding Modified Acetohydroxy Acid Synthasein which an Amino Acid is Substituted with Another Amino Acid at theSame Position

In the modified acetohydroxy acid synthase proteins confirmed in Example5, to confirm the effects of position in modification, vectors wereprepared in which the 96^(th) amino acid is substituted with an aminoacid other than threonine or serine, and the 503^(rd) amino acid issubstituted with an amino acid other than tryptophan or glutamine.

Specifically, overexpression vectors for L-valine biosynthesis, in whicha modification where the 503^(rd) amino acid of acetohydroxy acidsynthase is substituted with asparagine or leucine or a modificationwhere the 96^(th) amino acid is substituted with alanine or cysteine,were prepared from Corynebacterium glutamicum KCCM11201P, which is astrain producing L-valine. The substituted amino acids are only examplesof representative amino acids that can be substituted, and the aminoacids are not limited thereto.

For the preparation of these vectors, first, PCR was performed using thechromosomal DNA of Corynebacterium glutamicum KCCM11201P as a templateand a primer set of the primer 13 (SEQ ID NO: 18) and the primer 15 (SEQID NO: 20) and a primer set of the primer 16 (SEQ ID NO: 21) and theprimer 14 (SEQ ID NO: 19), and thereby an about 2,041 bp DNA fragmenthaving a BamHI restriction site at the 5′ end and a 1,055 bp DNAfragment having an XbaI restriction site at the 3′ end were amplified.PCR was performed as follows: denaturation at 94° C. for 5 min; 30cycles of denaturation at 94° C. for 30 sec, annealing at 56° C. for 30sec, and polymerization at 72° C. for 2 min; and polymerization at 72°C. for 7 min.

primer 15 (SEQ ID NO: 20): 5′-CTTCA TAGAA TAGGG TCTGG TTTTG GCGAA CCATGCCCAG-3′ primer 16 (SEQ ID NO: 21):5′-CTGGG CATGG TTCGC CAAAA CCAGA CCCTA TTCTA TGAAG-3′

Then, PCR was performed using the two amplified DNA fragments as atemplate and a primer set of the primer 13 (SEQ ID NO: 18) and theprimer 14 (SEQ ID NO: 19). PCR was performed as follows: denaturation at94° C. for 5 min; 30 cycles of denaturation at 94° C. for 30 sec,annealing at 56° C. for 30 sec, and polymerization at 72° C. for 4 min;and polymerization at 72° C. for 7 min.

As a result, an ilvBN gene fragment in which a modification where the503^(rd) amino acid of acetohydroxy acid synthase is substituted withasparagine was obtained.

In the same manner, PCR was performed using the chromosomal DNA ofCorynebacterium glutamicum KCCM11201P as a template and a primer set ofthe primer 13 (SEQ ID NO: 18) and the primer 17 (SEQ ID NO: 22) and aprimer set of the primer 18 (SEQ ID NO: 23) and the primer 14 (SEQ IDNO: 19), and thereby an about 2,041 bp DNA fragment having a BamHIrestriction site at the 5′ end and a 1,055 bp DNA fragment having anXbaI restriction site at the 3′ end were amplified.

primer 17 (SEQ ID NO: 22): 5′-CTTCA TAGAA TAGGG TCTGC AGTTG GCGAA CCATGCCCAG-3′ primer 18 (SEQ ID NO: 23):5′-CTGGG CATGG TTCGC CAACT GCAGA CCCTA TTCTA TGAAG-3′

Then, PCR was performed using the two amplified DNA fragments as atemplate and a primer set of the primer 13 (SEQ ID NO: 18) and theprimer 14 (SEQ ID NO: 19).

As a result, an ilvBN gene fragment in which a modification where the503^(rd) amino acid of acetohydroxy acid synthase is substituted withleucine was obtained.

In the same manner, PCR was performed using the chromosomal DNA ofCorynebacterium glutamicum KCCM11201P as a template and a primer set ofthe primer 13 (SEQ ID NO: 18) and the primer 19 (SEQ ID NO: 24) and aprimer set of the primer 20 (SEQ ID NO: 25) and the primer 14 (SEQ IDNO: 19), and thereby an about 819 bp DNA fragment having a BamHIrestriction site at the 5′ end and a 2,276 bp DNA fragment having anXbaI restriction site at the 3′ end were amplified.

primer 19 (SEQ ID NO: 24): 5′-GGTTG CGCCT GGGCC AGATG CTGCA ATGCA GACGCCAAC-3′ primer 20 (SEQ ID NO: 25):5′-GTTGG CGTCT GCATT GCAGC ATCTG GCCCA GGCGC AACC-3′

Then, PCR was performed using the two amplified DNA fragments as atemplate and a primer set of the primer 13 (SEQ ID NO: 18) and theprimer 14 (SEQ ID NO: 19).

As a result, an ilvBN gene fragment in which a modification where the96^(th) amino acid of acetohydroxy acid synthase is substituted withalanine was obtained.

In the same manner, PCR was performed using the chromosomal DNA ofCorynebacterium glutamicum KCCM11201P as a template and a primer set ofthe primer 13 (SEQ ID NO: 18) and the primer 21 (SEQ ID NO: 26) and aprimer set of the primer 22 (SEQ ID NO: 27) and the primer 14 (SEQ IDNO: 19), and thereby an about 819 bp DNA fragment having a BamHIrestriction site at the 5′ end and a 2,276 bp DNA fragment having anXbaI restriction site at the 3′ end were amplified.

primer 21 (SEQ ID NO: 26): 5′-GGTTG CGCCT GGGCC AGAGC ATGCA ATGCA GACGCCAAC-3′ primer 22 (SEQ ID NO: 27):5′-GTTGG CGTCT GCATT GCATG CTCTG GCCCA GGCGC AACC-3′

Then, PCR was performed using the two amplified DNA fragments as atemplate and a primer set of the primer 13 (SEQ ID NO: 18) and theprimer 14 (SEQ ID NO: 19).

As a result, an ilvBN gene fragment in which a modification where the96^(th) amino acid of acetohydroxy acid synthase is substituted withcysteine was obtained.

Using the same method as in Example 8, the four kinds PCR-amplifiedmodified gene fragments were treated with restriction enzymes BamHI andXbaI, and thereby the respective DNA fragments were obtained. These DNAfragments were each linked to the overexpression vector pECCG117 havingBamHI and XbaI restriction sites, transformed into E. coli DH5a, andplated on a solid LB medium containing kanamycin (25 mg/L).

The colonies transformed with a vector inserted with a target gene wereselected by PCR, and the plasmids were obtained by a commonly knownplasmid extraction method. These plasmid were each namedpECCG117-ilvB(W503N)N, pECCG117-ilvB(W503L)N, pECCG117-ilvB(T96A)N, andpECCG117-ilvB(T96C)N, each according to the sequence of modificationsinserted into the ilvB gene.

Example 10: Preparation of Strains in which Wild-Type-Derived ModifiedAcetohydroxy Acid Synthase is Introduced and Comparison ofL-Valine-Producing Abilities

The overexpression vectors for L-valine biosynthesis prepared inExamples 8 and 9 (i.e., pECCG117-ilvBN, pECCG117-ilvB(W503Q)N,pECCG117-ilvB(T96S)N and pECCG117-ilvB(W503N)N, pECCG117-ilvB(W503L)N,pECCG117-ilvB(T96A)N, and pECCG117-ilvB(T96C)N) were each inserted intothe wild-type Corynebacterium glutamicum strain (ATCC13032) byelectroporation. The prepared strains were each named Corynebacteriumglutamicum ATCC13032::pECCG117-ilvBN, Corynebacterium glutamicumATCC13032::pECCG117-ilvB (W503 Q)N, Corynebacterium glutamicumATCC13032::pECCG117-ilvB(T96S)N, Corynebacterium glutamicumATCC13032::pECCG117-ilvB(W503N)N, Corynebacterium glutamicumATCC13032::pECCG117-ilvB(W503L)N, Corynebacterium glutamicumATCC13032::pECCG117-ilvB(T96A)N, and Corynebacterium glutamicumATCC13032::pECCG117-ilvB(T96C)N.

Since those strains which are transformed with these vectors will beprovided with kanamycin resistance, the presence of transformation wasconfirmed by checking the growth of these strains in a medium containingkanamycin at a concentration of 25 mg/L.

Each of the strains was inoculated into a 250 mL corner-baffle flaskcontaining 25 mL of the production medium and cultured with shaking at200 rpm at 30° C. for 72 hours. The concentration of L-valine in eachculture was analyzed by HPLC (Table 4).

TABLE 4 Concentration of L-valine Production by Strains in WhichWild-Type-Derived Modified Acetohydroxy Acid Synthase is IntroducedL-Valine (g/L) Batch Batch Batch Strain 1 2 3 Mean ControlATCC13032::pECCG117-ilvBN 0.1 0.1 0   0.1 1 ATCC13032::pECCG117- 0.8 0.80.7 0.8 ilvB(W503Q)N 2 ATCC13032::pECCG117-ilvB(T96S)N 0.4 0.5 0.5 0.5 3ATCC13032::pECCG117- 0.7 0.6 0.5 0.6 ilvB(W503N)N 4 ATCC13032::pECCG117-0.7 0.7 0.5 0.5 ilvB(W503L)N 5 ATCC13032::pECCG117-ilvB(T96A)N 0.2 0.30.2 0.2 6 ATCC13032::pECCG117-ilvB(T96C)N 0.4 0.3 0.5 0.4

As a result, it was confirmed that the novel modifications in which the96^(th) or 503^(rd) amino acid of acetohydroxy acid synthase issubstituted with another amino acid showed a maximum increase of 700% inthe L-valine-producing ability compared to the control group. Thisresult confirmed the importance of the 96^(th) and 503^(rd) amino acidpositions of acetohydroxy acid synthase, and these amino acid positionsare expected to affect the ability of producing other branched-chainamino acids as well as L-valine.

Example 11: Preparation of Strains in which Modified Acetohydroxy AcidSynthase is Introduced and Comparison of L-Valine-Producing Abilities

To confirm whether the acetohydroxy acid synthase large subunit variantsof the present disclosure have an influence on the increase in theability of producing other L-branched-chain amino acids, as anotherembodiment of the L-branched-chain amino acids, the ability of producingL-leucine was examined.

Specifically, the two vectors in which novel modifications wereintroduced prepared in Example 6 were each transformed by a two-stephomologous recombination into the Corynebacterium glutamicum KCCM11661P(Korean Patent Application No. 10-2015-0119785 and Korean PatentApplication Publication No. 10-2017-0024653), which is anL-leucine-producing strain. Then, the strains in which the ilvB genemodification is introduced on the chromosome thereof were selected bynucleotide sequence analysis, and the strains in which the ilvB genemodification is introduced were named KCCM11661P::ilvB(W503Q) andKCCM11661P::ilvB(T96S).

The Corynebacterium glutamicum KCCM11661P having resistance tonorleucine (NL) is a mutant strain derived from Corynebacteriumglutamicum ATCC 14067 and was obtained as follows.

Specifically, the Corynebacterium glutamicum ATCC 14067 was cultured inan activation medium for 16 hours, and the activated strain wasinoculated into a seed medium, which was sterilized at 121° C. for 5minutes, and cultured for 14 hours, and 5 mL of the culture wasrecovered. The recovered culture was washed with 100 mM citric acidbuffer and N-methyl-N′-nitro-N-nitrosoguanidine (NTG) was added theretoto a final concentration of 200 mg/L and treated for 20 minutes, andwashed with 100 mM phosphate buffer. The strains treated with NTG wereplated on a minimal medium and the death rate was calculated, and as aresult, the death rate was shown to be 85%.

To obtain a mutant strain having resistance to norleucine (NL), theNTG-treated strains were plated on a minimal medium containing NL at afinal concentration of 20 mM, 30 mM, 40 mM, and 50 mM, respectively.Then, the strains were cultured at 30° C. for 5 days, and thereby anNL-resistant mutant strain was obtained.

<Activation Medium>

Meat Juice (1%), Polypeptone (1%), NaCl (0.5%), Yeast Extract (1%), Agar(2%), pH 7.2

<Seed Medium>

Glucose (5%), Bactopeptone (1%), NaCl (0.25%), Yeast Extract (1%), Urea(0.4%), pH 7.2

<Minimal Medium>

Glucose (1%), Ammonium Sulfate (0.4%), Magnesium Sulfate (0.04%),Monopotassium Phosphate (0.1%), Urea (0.1%), Thiamine (0.001%), Biotin(200 μg/L), Agar (2%), pH 7.0

The thus-obtained mutant strain was named Corynebacterium glutamicumKCJ-24 and deposited at the Korean Culture Center of Microorganisms(KCCM), which is recognized as an international depositary authorityunder the Budapest Treaty, on Jan. 22, 2015, under the Accession NumberKCCM11661P.

The KCCM11661P::ilvB(W503Q) and KCCM11661P::ilvB(T96S) were cultured inthe same manner as in Example 4, and the L-leucine concentration in eachculture therefrom was analyzed (Table 5).

TABLE 5 Concentration of L-Leucine Production by Strains in WhichKCCM11661P-Dervied Modified Acetohydroxy Acid Synthase is Introduced(g/L) Strain Batch 1 Batch 2 Batch 3 Mean Control KCCM11661P 2.7 2.6 2.92.7 1 KCCM11661P:: 3.1 3.3 3.3 3.2 ilvB(W503Q) 2 KCCM11661PP:: 3.0 3.23.1 3.1 ilvB(T96S)

The two strains in which novel modifications were introduced (i.e.,KCCM11661P::ilvB(W503Q) and KCCM11661P::ilvB(T96S)) showed a maximumincrease of 26.9% in the L-leucine-producing ability compared to theirparent strain.

Example 12: Preparation of Strains in which KCCM11662P-Derived ModifiedAcetohydroxy Acid Synthase is Introduced and Comparison ofL-Leucine-Producing Abilities

The two vectors in which novel modifications were introduced prepared inExample 6 were each transformed by a two-step homologous recombinationinto the Corynebacterium glutamicum KCCM11662P (Korean PatentApplication No. 10-2015-0119785 and Korean Patent ApplicationPublication No. 10-2017-0024653), which is an L-leucine-producingstrain. Then, the strains in which the ilvB gene modification isintroduced on the chromosome thereof were selected by nucleotidesequence analysis, and the strains in which the ilvB gene modificationis introduced were named KCCM11662P::ilvB(W503Q) andKCCM11662P::ilvB(T96S).

The Corynebacterium glutamicum KCCM11662P having resistance tonorleucine (NL) is a mutant strain derived from Corynebacteriumglutamicum ATCC 13869 and was obtained as follows.

Specifically, using Corynebacterium glutamicum ATCC 13869 as the parentstrain, the strain was cultured in the same manner for obtaining theKCCM11662P of Example 11 and finally an NL-resistant mutant strain wasobtained.

The thus-obtained mutant strain was named Corynebacterium glutamicumKCJ-28 and deposited at the Korean Culture Center of Microorganisms(KCCM), which is recognized as an international depositary authorityunder the Budapest Treaty, on Jan. 22, 2015, under the Accession NumberKCCM11662P.

The KCCM11662P::ilvB(W503Q) and KCCM11662P::ilvB(T96S) were cultured inthe same manner as in Example 4, and the L-leucine concentration in eachculture therefrom was analyzed (Table 6).

TABLE 6 Concentration of L-Leucine Production by Strains in WhichKCCM11662P-Dervied Modified Acetohydroxy Acid Synthase is Introduced(g/L) Strain Batch 1 Batch 2 Batch 3 Mean Control KCCM11662P 3.1 3.0 3.13.1 1 KCCM11662P:: 3.5 3.4 3.3 3.4 ilvB(W503Q) 2 KCCM11662PP:: 3.3 3.33.2 3.3 ilvB(T96S)

The two strains in which novel modifications were introduced (i.e.,KCCM11662P::ilvB(W503Q) and KCCM11662P::ilvB(T96S)) showed a maximumincrease of 13.3% in the L-leucine-producing ability compared to theirparent strain.

From the foregoing, a skilled person in the art to which the presentdisclosure pertains will be able to understand that the presentdisclosure may be embodied in other specific forms without modifying thetechnical concepts or essential characteristics of the presentdisclosure. In this regard, the exemplary embodiments disclosed hereinare only for illustrative purposes and should not be construed aslimiting the scope of the present disclosure. On the contrary, thepresent disclosure is intended to cover not only the exemplaryembodiments but also various alternatives, modifications, equivalents,and other embodiments that may be included within the spirit and scopeof the present disclosure as defined by the appended claims.

The invention claimed is:
 1. A polynucleotide encoding an acetohydroxyacid synthase variant having acetohydroxy acid synthase activity,wherein the acetohydroxy acid synthase variant comprises an acetohydroxyacid synthase large subunit (acetolactate synthase large subunit; IlvBprotein) having at least 95% sequence identity with the polypeptide ofSEQ ID NO: 1, wherein said large subunit comprises a substitution at theposition corresponding to position 96 of the polypeptide of SEQ ID NO:1, and wherein the amino acid at the position corresponding to position96 of the polypeptide of SEQ ID NO: 1 is a serine, cysteine, or alanine.2. The polynucleotide according to claim 1, wherein the acetohydroxyacid synthase large subunit further comprises a substitution at theposition corresponding to position 503 of the polypeptide of SEQ IDNO:1, and wherein the amino acid at the position corresponding toposition 503 of the polypeptide of SEQ ID NO:1 is a glutamine.
 3. Amicroorganism of the genus Corynebacterium producing an L-branched-chainamino acid, wherein the microorganism comprises an acetohydroxy acidsynthase variant or a polynucleotide encoding the acetohydroxy acidsynthase variant, wherein the acetohydroxy acid synthase variantcomprises an acetohydroxy acid synthase large subunit (acetolactatesynthase large subunit; IlvB protein) having at least 95% sequenceidentity with the polypeptide of SEQ ID NO: 1, wherein said largesubunit comprises a substitution at the position corresponding toposition 96 of the polypeptide of SEQ ID NO: 1, and wherein the aminoacid at the position corresponding to position 96 of the polypeptide ofSEQ ID NO: 1 is a serine, cysteine, or alanine.
 4. The microorganismaccording to claim 3, wherein the acetohydroxy acid synthase largesubunit further comprises a substitution at the position correspondingto position 503 of the polypeptide of SEQ ID NO:1, and wherein the aminoacid at the position corresponding to position 503 of the polypeptide ofSEQ ID NO:1 is a glutamine.
 5. The microorganism according to claim 3,wherein the microorganism of the genus Corynebacterium isCorynebacterium glutamicum.
 6. The microorganism according to claim 3,wherein the L-branched-chain amino acid is L-valine or L-leucine.